Method For Promoting Differentiation Of Pluripotent Stem Cells By Reducing Undifferentiated State Thereof

ABSTRACT

In related-art methods of differentiating pluripotent stem cells into a desired cell type, there has not been established a differentiation induction method using human ES/iPS cells and being highly efficient. Many attempts have been made, including a stepwise differentiation induction method based on the control of culture conditions or the addition of, for example, various cell growth factors/differentiation factors to a culture solution, but the use of complicated culture steps is a big problem. A method of inducing differentiation into a desired cell type within a short period of time and with high efficiency by use of a pluripotent stem cell that actively undergoes cell differentiation, which is obtained by reducing an undifferentiated state of the pluripotent stem cell, has been developed, and thus the present invention has been completed.

TECHNICAL FIELD

The present invention relates to a method of reducing anundifferentiated state of a pluripotent stem cell, and morespecifically, to a method of differentiating a pluripotent stem cellinto a desired cell type with high efficiency and a differentiationinduction kit to be used for the differentiation method.

The present application claims priority from Japanese Patent ApplicationNo. 2016-016785, which is incorporated herein by reference.

BACKGROUND ART

(On Induction of Differentiation of Pluripotent Stem Cells)

Regenerative medicine using cells obtained by inducing differentiationof pluripotent stem cells, such as embryonic stem cells (ES cells) orinduced pluripotent stem cells (iPS cells) is a therapeutic method forwhich all peoples of the world have high expectations and which aredesired to be realized soon. As regenerative medicine, a transplantationtherapy with retinal pigment epithelial cells derived from iPS cells isfresh in our memory. However, a technology for rapidly generating maturedifferentiated cells suited for cell transplantation in a sufficientamount is still under development and has much room for development.

A current mainstream method of inducing differentiation of pluripotentstem cells into a desired cell type is a method involving sequentiallyadding cytokines/growth factors suited for respective differentiationstages to a medium to cause differentiation via an embryoid body andprogenitor cells. This method has problems in, for example, that aculture period until differentiated cells of interest are obtained islong, that differentiation induction efficiency is not high, and thatcells of different cell lineages are mixed with each other.

In recent years, attempts have been actively made to direct celldifferentiation by forcibly expressing, in ES/iPS cells, one or acombination of a plurality of tissue-specifically expressedtranscription factors. This differentiation induction method usingtranscription factors can directly induce ES/iPS cells intodifferentiated cells of interest, and hence is expected to be extremelyeffective means. However, even with this technique, differentiationinduction efficiency for some cell types is still low, and thus, it isdifficult to obtain a sufficient number of differentiated cells requiredfor regenerative medicine.

In view of the foregoing, there has been a demand for development of anovel differentiation induction method for producing differentiatedcells of interest from pluripotent stem cells more rapidly and moreuniformly with higher efficiency.

(Current Situation of Induction of Differentiation of Pluripotent StemCells in Related Art)

Non Patent Literatures 1 to 4, which are related art, are each directedto a system for facilitating induction of differentiation of ES/iPScells. For an example, there was a disclosure that ES/iPS cells wereinduced into skeletal muscle differentiation.

However, those differentiation induction methods are clearly differentfrom a differentiation induction method of the present invention.

In Non Patent Literature 5, there was a disclosure that “in researchusing mouse ES cells, when expression of a transcription factor Oct3/4is repressed, trophectoderm can be derived from the ES cells.” Inaddition, in Patent Literature 1, there was a disclosure that“differentiation of neural stem cells into neurons, glial cells, and thelike can be regulated by controlling an expression amount of an Oct-3/4protein in the neural stem cells.”

However, there was no disclosure or suggestion of a method ofefficiently inducing differentiation into a desired cell type byintroducing a transcription factor required for induction ofdifferentiation into the desired cell type into a pluripotent stem cell.

CITATION LIST Patent Literature

-   [PTL 1] JP 2004-236607 A

Non Patent Literature

-   [NPL 1] Nature medicine 13: 642-648.-   [NPL 2] Cell stem cell 10: 610-619.-   [NPL 3] Mol Ther. November; 20(11): 2153-67.-   [NPL 4] PLoS One. 2013 Apr. 23; 8(4): e61540.-   [NPL 5] Nat. Genet. 24(4): 372-6.

SUMMARY OF INVENTION Technical Problem

In related-art methods of differentiating pluripotent stem cells into adesired cell type, there has not been established a differentiationinduction method using human ES/iPS cells and being stable and highlyefficient. Many attempts have been made, including a stepwisedifferentiation induction method based on the control of cultureconditions or the addition of, for example, various cell growthfactors/differentiation factors to a culture solution, but the use ofcomplicated culture steps is a big problem. In addition, there are alsobig problems in, for example, that the speed of cell differentiation islow, and hence long-period culture is required, and that thedifferentiation efficiency is low, and hence it is difficult to obtain asufficient number of required cells.

Solution to Problem

The inventors of the present invention have presumed that theabove-mentioned problems are partly due to the fact that pluripotentstem cells have a property of maintaining the undifferentiated state ofthe cells by various mechanisms. In view of this, the inventors havedeveloped a method of inducing differentiation into a desired cell typewithin a short period of time and with high efficiency by reducingundifferentiated state maintenance of a pluripotent stem cell togenerate a pluripotent stem cell that actively proceeds to adifferentiated cell type.

Further, the inventors have developed a method of more potently inducingdifferentiation into a desired cell type within a short period of timeand with high efficiency by not only reducing undifferentiated statemaintenance of a pluripotent stem cell but also reducing differentiationresistance thereof.

Thus, the present invention has been completed.

That is, the present invention includes the following.

1. A differentiation induction kit for differentiating a pluripotentstem cell into a desired cell type, including at least any one of thefollowing items (1) to (8):

(1) a pluripotent stem cell and a gene for a compound having an actionof substantially removing or reducing an expression amount of a POU5F1protein;

(2) a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced;

(3) a pluripotent stem cell in which a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein is forcibly expressed;

(4) a gene construct carrying a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, and a pluripotent stem cell;

(5) a pluripotent stem cell having a gene construct carrying a gene fora compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein inserted into a genome thereof;

(6) a pluripotent stem cell in which a POU5F1 gene has been disrupted;

(7) a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced and which has ahistone in which H3K27me3 modification has been substantially removed orreduced; and

(8) a pluripotent stem cell, and a gene for a compound having an actionof substantially removing or reducing an expression amount of a POU5F1protein and a demethylase gene.

2. A differentiation induction kit according to the above-mentioned item1, wherein the gene for the compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein includes agene expressing siRNA of POU5F1, a gene expressing shRNA of POU5F1, agene expressing an antisense strand of POU5F1, and/or a gene for anantibody against POU5F1.

3. A differentiation induction kit according to the above-mentioned item1 or 2, further including a transcription factor required for inductionof differentiation into the desired cell type.

4. A differentiation induction kit according to the above-mentioned item3, wherein a transcription factor gene for the transcription factor iscarried on a Sendai virus vector.

5. A differentiation induction kit for differentiating a pluripotentstem cell into a skeletal muscle cell, including at least any one of thefollowing items (1) to (8):

(1) a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced, and a transcriptionfactor MYOD1;

(2) a gene for a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein, a transcriptionfactor MYOD1, and a pluripotent stem cell;

(3) a pluripotent stem cell in which a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein is forcibly expressed, and a transcription factor MYOD1;

(4) a gene construct carrying a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a transcription factor MYOD1, and a pluripotent stem cell;

(5) a pluripotent stem cell having a gene construct carrying a gene fora compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein inserted into a genome thereof,and a transcription factor MYOD1;

(6) a pluripotent stem cell in which a POU5F1 gene has been disrupted,and a transcription factor MYOD1;

(7) a transcription factor MYOD1, and a pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and which has a histone in which H3K27me3 modification hasbeen substantially removed or reduced; and

(8) a pluripotent stem cell, and a transcription factor MYOD1, a genefor a compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein, and a demethylase gene.

6. A differentiation induction kit for differentiating a pluripotentstem cell into a neuron, including at least any one of the followingitems (1) to (8):

(1) a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced, and transcriptionfactors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2;

(2) a gene for a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein, transcription factorsNEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2, and a pluripotent stemcell;

(3) a pluripotent stem cell in which a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein is forcibly expressed, and transcription factors NEUROG1,NEUROG2, NEUROG3, NEUROD1, and NEUROD2;

(4) a gene construct carrying a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, andNEUROD2, and a pluripotent stem cell;

(5) a pluripotent stem cell having a gene construct carrying a gene fora compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein inserted into a genome thereof,and transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, andNEUROD2;

(6) a pluripotent stem cell in which a POU5F1 gene has been disrupted,and transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, andNEUROD2;

(7) transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, andNEUROD2, and a pluripotent stem cell in which an expression amount of aPOU5F1 protein has been substantially removed or reduced and which has ahistone in which H3K27me3 modification has been substantially removed orreduced; and

(8) a pluripotent stem cell, and transcription factors NEUROG1, NEUROG2,NEUROG3, NEUROD1, and NEUROD2, a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, and a demethylase gene.

7. A differentiation induction kit for differentiating a pluripotentstem cell into a hepatocyte, including at least any one of the followingitems (1) to (8):

(1) a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced, and a transcriptionfactor HNF1A;

(2) a gene for a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein, a transcriptionfactor HNF1A, and a pluripotent stem cell;

(3) a pluripotent stem cell in which a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein is forcibly expressed, and a transcription factor HNF1A;

(4) a gene construct carrying a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a transcription factor HNF1A, and a pluripotent stem cell;

(5) a pluripotent stem cell having a gene construct carrying a gene fora compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein inserted into a genome thereof,and a transcription factor HNF1A;

(6) a pluripotent stem cell in which a POU5F1 gene has been disrupted,and a transcription factor HNF1A;

(7) a transcription factor HNF1A, and a pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and which has a histone in which H3K27me3 modification hasbeen substantially removed or reduced; and

(8) a pluripotent stem cell, a transcription factor HNF1A, a demethylasegene, and a gene for a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein.

8. A differentiation induction kit according to the above-mentioned item3 or 4, wherein the differentiation induction kit is as described in anyone of the following items (1) to (6):

(1) the transcription factor required for induction of differentiationinto the desired cell type includes MYOD1, and the desired cell typeincludes a skeletal muscle cell;

(2) the transcription factor required for induction of differentiationinto the desired cell type includes HNF1A, and the desired cell typeincludes a hepatocyte;

(3) the transcription factor required for induction of differentiationinto the desired cell type includes SOX9, and the desired cell typeincludes a chondrocyte;

(4) the transcription factor required for induction of differentiationinto the desired cell type includes RUNX2, and the desired cell typeincludes a bone cell;

(5) the transcription factor required for induction of differentiationinto the desired cell type includes SPI1, and the desired cell typeincludes a hematopoietic cell; and

(6) the transcription factor required for induction of differentiationinto the desired cell type includes ASCL1, and the desired cell typeincludes a neuron.

9. A method of differentiating a pluripotent stem cell into a desiredcell type, including any one of the following steps (1) to (9):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, and a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein, and a transcription factor gene required forinduction of differentiation into the desired cell type into a genome ofa pluripotent stem cell;

(3) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein, and a gene construct carrying atranscription factor required for induction of differentiation into thedesired cell type into a genome of a pluripotent stem cell;

(4) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell,in which an expression amount of a POU5F1 protein has been substantiallyremoved or reduced;

(5) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell,in which a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein is forcibly expressed;

(6) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein, and atranscription factor required for differentiation into the desired celltype to a pluripotent stem cell;

(7) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell,in which a POU5F1 gene has been disrupted;

(8) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and a transcription factor required forinduction of differentiation into the desired cell type to a pluripotentstem cell; and

(9) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell inwhich an expression amount of a POU5F1 protein has been substantiallyremoved or reduced and which has a histone in which H3K27me3modification has been substantially removed or reduced.

10. A differentiation method according to the above-mentioned item 9,wherein the gene for the compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein includes agene expressing siRNA of POU5F1, a gene expressing shRNA of POU5F1, agene expressing an antisense strand of POU5F1, and/or a gene for anantibody against POU5F1.

11. A differentiation method according to the above-mentioned item 9 or10, wherein a gene for the transcription factor is carried on a Sendaivirus vector.

12. A method of differentiating a pluripotent stem cell into a skeletalmuscle cell, including any one of the following steps (1) to (9):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and a transcription factor MYOD1 to a pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene for a transcription factor MYOD1into a genome of a pluripotent stem cell;

(3) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene construct carrying a transcriptionfactor MYOD1 into a genome of a pluripotent stem cell;

(4) a step of adding a transcription factor MYOD1 to a pluripotent stemcell in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(5) a step of adding a transcription factor MYOD1 to a pluripotent stemcell in which a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein is forcibly expressed;

(6) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein and atranscription factor MYOD1 to a pluripotent stem cell;

(7) a step of adding a transcription factor MYOD1 to a pluripotent stemcell in which a POU5F1 gene has been disrupted;

(8) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and a transcription factor MYOD1 to apluripotent stem cell; and

(9) a step of adding a transcription factor MYOD1 to a pluripotent stemcell in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

13. A method of differentiating a pluripotent stem cell into a neuron,including any one of the following steps (1) to (9):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1,and/or NEUROD2 to a pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and genes for transcription factors NEUROG1,NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 into a genome of a pluripotentstem cell;

(3) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene construct carrying transcriptionfactors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 into a genomeof a pluripotent stem cell;

(4) a step of adding transcription factors NEUROG1, NEUROG2, NEUROG3,NEUROD1, and/or NEUROD2 to a pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced;

(5) a step of adding transcription factors NEUROG1, NEUROG2, NEUROG3,NEUROD1, and/or NEUROD2 to a pluripotent stem cell in which a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein is forcibly expressed;

(6) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein andtranscription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2to a pluripotent stem cell;

(7) a step of adding transcription factors NEUROG1, NEUROG2, NEUROG3,NEUROD1, and/or NEUROD2 to a pluripotent stem cell in which a POU5F1gene has been disrupted;

(8) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and transcription factors NEUROG1, NEUROG2,NEUROG3, NEUROD1, and/or NEUROD2 to a pluripotent stem cell; and

(9) a step of adding transcription factors NEUROG1, NEUROG2, NEUROG3,NEUROD1, and/or NEUROD2 to a pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and which has a histone in which H3K27me3 modification has beensubstantially removed or reduced.

14. A method of differentiating a pluripotent stem cell into ahepatocyte, including any one of the following steps (1) to (9):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and a transcription factor HNF1A to a pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene for a transcription factor HNF1Ainto a genome of a pluripotent stem cell;

(3) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene construct carrying a transcriptionfactor HNF1A into a genome of a pluripotent stem cell;

(4) a step of adding a transcription factor HNF1A to a pluripotent stemcell in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(5) a step of adding a transcription factor HNF1A to a pluripotent stemcell in which a compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein is forcibly expressed;

(6) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein and atranscription factor HNF1A to a pluripotent stem cell;

(7) a step of adding a transcription factor HNF1A to a pluripotent stemcell in which a POU5F1 gene has been disrupted;

(8) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and a transcription factor HNF1A to apluripotent stem cell; and

(9) a step of adding a transcription factor HNF1A to a pluripotent stemcell in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

15. A differentiation method according to any one of the above-mentioneditems 9 to 11, wherein the differentiation method is as described in anyone of the following items (1) to (6):

(1) the transcription factor required for induction of differentiationinto the desired cell type includes MYOD1, and the desired cell typeincludes a skeletal muscle cell;

(2) the transcription factor required for induction of differentiationinto the desired cell type includes HNF1A, and the desired cell typeincludes a hepatocyte;

(3) the transcription factor required for induction of differentiationinto the desired cell type includes SOX9, and the desired cell typeincludes a chondrocyte;

(4) the transcription factor required for induction of differentiationinto the desired cell type includes RUNX2, and the desired cell typeincludes a bone cell;

(5) the transcription factor required for induction of differentiationinto the desired cell type includes SPI1, and the desired cell typeincludes a hematopoietic cell; and

(6) the transcription factor required for induction of differentiationinto the desired cell type includes ASCL1, and the desired cell typeincludes a neuron.

Advantageous Effects of Invention

The method of differentiating a pluripotent stem cell into a desiredcell type with high efficiency and differentiation induction kit fordifferentiating a pluripotent stem cell into a desired cell type withhigh efficiency of the present invention each have at least any one ormore of the following effects.

(1) The period of time required for cell differentiation starting withthe pluripotent stem cell is shortened and the differentiation inductionefficiency is improved.(2) As modified synthetic mRNA for a gene is used to introduce the geneinto the pluripotent stem cell, the introduced gene is not incorporatedinto the genome of the pluripotent stem cell, and the result is thatthere is no risk of canceration or the like after cell differentiationinduction.(3) In the introduction of the gene into the pluripotent stem cell usingthe modified synthetic mRNA, the timing and number of times of theaddition of the mRNA for the gene can be easily changed, and henceoptimal conditions specific to each of cell types can be selected.(4) A method of reducing undifferentiated state maintenance of apluripotent stem cell and a method of reducing differentiationresistance thereof are combined with each other to shorten the period oftime required for cell differentiation starting with the pluripotentstem cell and improve the differentiation induction efficiency in asynergistic manner.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A schematic diagram of a method of inducing differentiation of apluripotent stem cell in which an expression amount of a POU5F1 proteinhas been reduced of the present invention. It is illustrated thatdifferentiation into desired cells can be induced by introducing(adding) modified synthetic RNAs for tissue-specific transcriptionfactors to pluripotent stem cells in which the function of POU5F1, agene responsible for an undifferentiated state, has been suppressed byan RNA interference method.

[FIGS. 2A-2D] FIG. 2A A schematic diagram of a method of reducingdifferentiation resistance of a pluripotent stem cell to a desired celltype of the present invention. FIG. 2B When H3K27me3 modification in ahuman ES or iPS cell is reduced or removed, a transcription factor (TF)binds to the promoter site of a downstream gene to enhance theexpression of a group of development/differentiation-related genes,resulting in differentiation. FIG. 2C A method of inducingdifferentiation of a human ES cell or iPS cell by introducing modifiedsynthetic mRNA for a demethylase, and then introducing modifiedsynthetic mRNA for the transcription factor (TF). FIG. 2D A method ofinducing differentiation of a human ES cell or iPS cell bysimultaneously introducing the modified synthetic mRNAs for thedemethylase and the transcription factor (TF).

FIG. 3 A schematic view of a differentiation induction method usingmodified synthetic mRNA for a target gene.

FIG. 4 A schematic view of a differentiation step using modifiedsynthetic mRNA for a target gene.

FIG. 5 A method of introducing a target gene into the genome of apluripotent stem cell.

FIG. 6 The suppression of the expression of the POU5F1 protein by siRNA.Human ES cells were transfected with siRNA of POU5F1 (siPOU5F1), and itwas confirmed by western blot using a specific antibody that the proteinof POU5F1 was decreased (left figures) and it was confirmed by animmunostaining method using a specific antibody that the proteins ofPOU5F1 and NANOG were decreased (right figures). As a control, scramblesiRNA (siControl) not acting on any gene was used. β-ACTIN represents a“loading control”, and DAPI represents “cell nuclei”. [FIG. 7] Themorphological change of pluripotent stem cells by siPOU5F1. It is shownthat human ES cells changed into flat shapes when transfected withsiPOU5F1.

FIG. 8 It is shown that the co-transfection of siPOU5F1 andtranscription factor mRNA activates differentiation genes. It wasconfirmed by a real-time PCR method that the transfection of human EScells with modified synthetic RNAs (synRNAs) for tissue-specifictranscription factors (MYOD1, HNF1A, RUNX2, SOX9, SPI1, and ASCL1) incombination with siPOU5F1 increased the expression of respectivedifferentiation marker genes (MYOG, AFP, COL1A1, COL2A1, CD45, andNESTIN). Right bars represent cases of transfection with siPOU5F1, andleft bars represent cases of transfection with siControl. mCherry orEmerald was used as synRNA serving as a negative control.

FIG. 9 Skeletal muscle differentiation by siPOU5F1 and MYOD1-synRNA wasconfirmed by an immunostaining method using an MyHC antibody. It wasconfirmed that, although MYOD1 alone (siControl+MYOD1) hardly causedmuscle differentiation, its combination with siPOU5F1 dramaticallyinduced muscle differentiation. MyHC represents a “terminaldifferentiation marker”, and DAPI represents “cell nuclei”.

FIG. 10 Skeletal muscle differentiation in more than one ES cell linesand iPS cell lines by siPOU5F1 and MYOD1-synRNA was confirmed by animmunostaining method using an MyHC antibody. The co-expression ofsiPOU5F1 and MYOD1-synRNA was able to induce skeletal muscledifferentiation rapidly and with high efficiency also in an H9 lineserving as an ES cell line, and four kinds of iPS cell lines (201B7,409B2, RIKEN-1A, and tkDA3-4).

FIG. 11 Hepatocyte differentiation by siPOU5F1 and HNF1A-synRNA is shownby an immunostaining method using an albumin antibody and an AFPantibody. DAPI represents “cell nuclei”.

FIG. 12 It was confirmed by a real-time PCR method that differentiationinduction by siPOU5F1 was further facilitated by combining forcedexpression of a demethylase therewith. It was confirmed that theexpression of a differentiation marker gene (MYOG, AFP, COL2A, or CD45)activated by siPOU5F1 anda tissue-specific transcription factor (MYOD1,HNF1A, SOX9, or SPI1) was further upregulated by forced expression ofthe demethylase in a synergistic manner.

DESCRIPTION OF EMBODIMENTS

A method of reducing an undifferentiated state of a pluripotent stemcell of the present invention (hereinafter sometimes referred to as“method of the present invention”) is described below, though the methodis not particularly limited as long as the method is a method capable ofreducing undifferentiated state maintenance of a pluripotent stem cell,and further, is a method capable of not only reducing undifferentiatedstate maintenance of a pluripotent stem cell but also reducingdifferentiation resistance thereof as required.

(Pluripotent Stem Cell)

The pluripotent stem cell to be used in the method of the presentinvention is not particularly limited, but is preferably derived from amammal, particularly preferably derived from a human. The pluripotentstem cell is, for example, a human ES cell, a human iPS cell, or anycombination thereof, is not particularly limited, and encompasses tissuestem cells derived from tissues and organs, dermal fibroblasts, and allkinds of cells derived from tissues or organs.

(Reducing Undifferentiated State Maintenance of Pluripotent Stem Cell)

In pluripotent stem cells, the expression of a transcription factorPOU5F1 (SEQ ID NOS: 1 and 2: POU domain, class 5, transcription factor 1isoform 1: http://www.ncbi.nlm.nih.gov/protein/NP_002692, other names:OCT3, OCT4, OTF3, OTF4, OTF-3, Oct-3, Oct-4, MGC22487) is essential tothe undifferentiated state maintenance of the pluripotent stem cells.POU5F1 is specifically expressed in pluripotent cells, such asreproductive cells and a preimplantation early embryo. In Examples ofthe present invention, it has been confirmed that differentiation into adesired cell type can be efficiently induced by introducing atranscription factor required for induction of differentiation into thedesired cell type into a pluripotent stem cell in which an expressionamount of a POU5F1 protein has been reduced (see FIG. 1). That is, the“reducing undifferentiated state maintenance of a pluripotent stem cell”of the present invention means substantially removing or reducing anexpression amount of a POU5F1 protein in the pluripotent stem cell. Thesubstantially removing or reducing an expression amount of a POU5F1protein encompasses inhibiting the process of any one of thetranscription and translation stages of POU5F1 and/or inhibiting theactivity of the translated POU5F1 protein, and is not particularlylimited.

In addition, a state in which the expression amount of the POU5F1protein in the pluripotent stem cell has been substantially removed orreduced may be confirmed by a comparison to the degree of the expressionamount of the POU5F1 protein (or expression amount of the POU5F1 gene)in a pluripotent stem cell that has not been subjected to the removingor the reducing. For example, the state (degree) in which the expressionamount of the POU5F1 protein in the pluripotent stem cell has beensubstantially removed or reduced is from 95 to 1, from 90 to 2, from 85to 3, from 80 to 4, from 75 to 5, from 70 to 6, from 65 to 7, from 60 to8, from 50 to 10, from 40 to 15, from 30 to 20, or about 25 whencompared to the expression amount of the POU5F1 protein in thepluripotent stem cell that has not been removed or reduced, which isdefined as 100. The degree of the expression amount of the POU5F1protein in the pluripotent stem cell may be easily measured by using acommercially available anti-POU5F1 antibody, and the gene expressionamount of POU5F1 may be measured by a method known per se.

(Reducing Differentiation Resistance of Pluripotent Stem Cell to DesiredCell Type)

In pluripotent stem cells, a special chromatin structure called a“bivalent domain” is formed in each promoter region of a group of genesinvolved in differentiation, and under a stemness-maintaining state, thegroup of genes involved in development/differentiation are in a standbystate so as not to be easily expressed. The inventors of the presentinvention have confirmed that “as a methyl group modification of ahistone called H3K27me3 is removed or reduced in the “bivalent domain”,the expression of differentiation genes required for induction ofdifferentiation into the desired cell type is rapidly and efficientlyfacilitate& (see FIGS. 2A-2D).

Namely, the “reducing differentiation resistance of a pluripotent stemcell to a desired cell type” of the present invention means that theH3K27me3 modification of the pluripotent stem cell is substantiallyremoved or reduced.

In addition, a state in which the H3K27me3 modification of thepluripotent stem cell has been substantially removed or reduced may beconfirmed by a comparison to the degree of the H3K27me3 modification ofa pluripotent stem cell that has not been subjected to the removing orthe reducing. For example, the state (degree) in which the H3K27me3modification of the pluripotent stem cell has been substantially removedor reduced is from 95 to 1, from 90 to 2, from 85 to 3, from 80 to 4,from 75 to 5, from 70 to 6, from 65 to 7, from 60 to 8, from 50 to 10,from 40 to 20, or about 30 when compared to the degree of the H3K27me3modification of the pluripotent stem cell that has not been removed orreduced, which is defined as 100. The degree of the H3K27me3modification of the pluripotent stem cell may be easily measured byusing a commercially available anti-Histone H3K27me3 antibody, and thegene expression amount of H3K27me3 may be measured by a method known perse.

(Method of inducing Differentiation of Pluripotent Stem Cell intoDesired Cell Type with High Efficiency of the Present Invention)

As described above, the method of the present invention is notparticularly limited as long as the method is a method of reducingundifferentiated state maintenance of a pluripotent stem cell, andfurther, is a method capable of reducing differentiation resistance of apluripotent stem cell to the desired cell type as required, and may beexemplified by the following.

(Use of Modified Synthetic mRNA for Target Gene)

The method of the present invention includes adding (introducing,transfecting), to a pluripotent stem cell, a gene fora compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein (gene expressing small interfering RNA (siRNA) againstPOU5F1, gene expressing shRNA against POU5F1, gene expressing anantisense strand of POU5F1, or antibody gene), and further, a gene for atranscription factor required for induction of differentiation into thedesired cell type.

Similarly, the method of the present invention includes adding(introducing, transfecting), to a pluripotent stem cell, a gene for acompound having an action of substantially removing or reducing H3K27me3modification as required, and further, a gene for a transcription factorrequired for induction of differentiation of the pluripotent stem cellinto the desired cell type.

The term “gene” as used herein encompasses not only double-strandednucleic acids, but also their respective constituent single strands,such as plus strands (or sense strands) or complementary strands (orantisense strands), linear nucleic acids, and circular nucleic acids,and encompasses DNA, RNA, mRNA, cDNA, and the like, unless otherwisestated.

In addition, the term “target gene” is meant to encompass both of: thegene for the compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein and/or the gene forthe compound having an action of substantially removing or reducingH3K27me3 modification; and the transcription factor required forinduction of differentiation into the desired cell type.

In a step of the method of the present invention, a method known per semay be used without any particular limitation as a method of adding(introducing), to the pluripotent stem cell, the gene for the compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein (or the gene for the compound having anaction of substantially removing or reducing H3K27me3 modification)and/or the transcription factor required for induction ofdifferentiation into the desired cell type. There is preferably used amethod of inducing differentiation by efficiently introducing syntheticmRNA for a transcription factor into human pluripotent stem cellsthrough use of a gene expression method involving using synthetic mRNAdeveloped by Warren, Rossi, et al. (reference: Cell Stem Cell 7:618-630, 2010.), which is a footprint-free forced gene expression methodcausing no gene incorporation into a host genome (see FIG. 3).

The timing at which the gene for the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the gene for the compound having an action of substantiallyremoving or reducing H3K27me3 modification) and the transcription factorrequired for induction of differentiation into the desired cell type areadded to the pluripotent stem cell is not particularly limited, but itis preferred that the gene for the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the gene for the compound having an action of substantiallyremoving or reducing H3K27me3 modification) be added to the pluripotentstem cell before the addition of the transcription factor required fordifferentiation induction.

Further, with regard to the addition timing of each gene (mRNA), theaddition may be performed, for example, one or more times, preferablytwo to five times, two to four times, two or three times, or two timesevery 12 hours to 64 hours, but the addition timing is not particularlylimited thereto. A more specific method may be exemplified by thefollowing.

(Synthesis of Modified mRNA Encoding Amino Acid Sequence ofTranscription Factor)

Modified mRNA is synthesized with a referred method described in theliterature “Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5): 618-30.”More specifically, mRNA is synthesized by in vitro transcription using amixture of dNTPs {(dNTPs: 3-O-Me-m⁷G (5′) ppp (5′)G ARCA cap analog,5-methylcytidine triphosphate, and pseudouridine triphosphate)} obtainedby modifying template DNA encoding the amino acid sequence of thetranscription factor required for induction of differentiation into thedesired cell type.

(Generation of Sendai Virus Vector encoding Amino Acid Sequence ofTranscription Factor)

In order to express a mammalian (in particular, human) transcriptionfactor, a Sendai virus vector capable of expressing a humantranscription factor is preferably used. In particular, a mutant of aSendai virus vector, such as an F protein-deficient mutant, has noinfectivity, and is easy to handle (see Inoue et al., J Virol. 77:23238-3246, 2003).

(Method of Inducing Differentiation of Pluripotent Stem Cell intoDesired Cell Type with High Efficiency)

A single transcription factor or a cocktail of two or more transcriptionfactors required for induction of differentiation into the desired celltype is prepared. The form of the transcription factors is notparticularly limited, and may be any of synthetic mRNAs, a Sendai virusvector having incorporated therein a transcription factor (or aplurality of transcription factors), and nanoparticle capsulescontaining synthetic mRNAs.

A method of introducing the single transcription factor or cocktail oftwo or more transcription factors described above into cells is notparticularly limited, and transfection with Lipofectamine, viralinfection, or the like is utilized. A schematic view of one example ofthe differentiation induction step that may be utilized in the method ofthe present invention is illustrated in FIG. 4.

(Use of Expression Vector)

In a step of the method of the present invention, an expression vectorknown per se having introduced therein the gene for the compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein (or the gene for the compound having an action ofsubstantially removing or reducing H3K27me3 modification) and/or thetranscription factor required for induction of differentiation into thedesired cell type may be used. Examples of the expression vector to beused in the present invention may include, but not particularly limitedto, an animal cell expression plasmid vector, a Sendai virus vector andothers.

A method of introducing the synthetic mRNA and the expression vectorinto the pluripotent stem cell is not particularly limited, but examplesthereof may include a lipofection method, a liposome method, anelectroporation method, a calcium phosphate coprecipitation method, adiethylaminoethyl (DEAE)-dextran method, a microinjection method, a genegun method and others. A particularly preferred example is a lipofectionmethod.

Another method may involve using an expression vector for the gene forthe compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein (or the gene for the compoundhaving an action of substantially removing or reducing H3K27me3modification), and using synthetic mRNA for the transcription factorrequired for induction of differentiation into the desired cell type, ormay adopt the opposite pattern.

(Compound Having Action of Substantially Removing or Reducing ExpressionAmount of POU5F1 Protein)

The compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein of the present invention is notparticularly limited, but is, for example, siRNA against POU5F1, shRNAagainst POU5F1, an antisense strand of POU5F1, an antibody thatspecifically binds to the POU5F1 protein, or an inhibitor.

In addition, not only by using those compounds alone, but also by usinga plurality of kinds of compounds and/or a low-molecular-weight compoundin combination, it is possible to efficiently “reduce anundifferentiated state of a pluripotent stem cell (substantially removeor reduce an expression amount of a POU5F1 protein in a pluripotent stemcell).”

(Compound Having Action of Substantially Removing or Reducing H3K27Me3Modification)

The compound having an action of substantially removing or reducingH3K27me3 modification of the present invention is not particularlylimited, but is, for example, a demethylase (in particular, ademethylase having an action of removing a methyl group of H3K27me3), anantibody that specifically binds to H3K27me3, an antibody forPolycomb-group proteins (PcG proteins) having an H3K27me3 modificationaction, siRNA (in particular, cationic siRNA), or an inhibitor. Thecationic siRNA does not require a reagent for transfection.

Examples of the low-molecular-weight compound may include, but notparticularly limited to, histone deaceylase (HDAC) inhibitors, such asvalproic acid.

Examples of the demethylase include AOF (LSD1), AOF1 (LSD2), FBXL11(JHDM1A), Fbx110 (JHDM1B), FBXL19 (JHDM1C), KIAA1718 (JHDM1D), PHF2(JHDM1E), PHF8 (JHDM1F), JMJD1A (JHDM2A), JMJD1B (JHDM2B), JMJD1C(JHDM2C), JMJD2A (JHDM3A), JMJD2B (JHDM3B), JMJD2C (JHDM3C), JMJD2D(JHDM3D), RBP2 (JARID1A), PLU1 (JARID1B), SMCX (JARID1C), SMCY(JARID1D), Jumonji (JARID2), UTX (UTX), UTY (UTY), JMJD3 (JMJD3), JMJD4(JMJD4), JMJD5 (JMJD5), JMJD6 (JMJD6), JMJD7 (JMJD7), and JMJD8 (JMJD8).Of those, JMJD3, UTX, or the like is preferred as a demethylase havingan action of removing a methyl group of H3K27me3.

In addition, the demethylase of the present invention also includes thefollowing:

(1) a protected derivative, sugar chain-modified derivative, acylatedderivative, or acetylated derivative of any one of the demethylasesdescribed above;

(2) an enzyme that has 90% (or 92%, 94%, 96%, 98%, or 99%) or morehomology to any one of the demethylases described above and has asubstantially equivalent action of substantially removing or reducingH3K27me3 modification to that of the demethylase; and

(3) an enzyme that has 100 to 10, 50 to 30, 40 to 20, 10 to 5, or 5 to 1amino acid substituted, deleted, inserted, and/or added in any one ofthe demethylases described above and has a substantially equivalentaction of substantially removing or reducing H3K27me3 modification tothat of the demethylase.

Further, the gene of the demethylase of the present invention includesthe following:

(1) a gene encoding a polypeptide formed of the amino acid sequence ofany one or more of the enzymes described above;

(2) a gene encoding a polypeptide that has 1 to 20 (or 1 to 15, 1 to 10,1 to 7, 1 to 5, or 1 to 3) amino acids substituted, deleted, inserted,and/or added in the amino acid sequence of any one or more of theenzymes described above and has a substantially equivalent action ofsubstantially removing or reducing H3K27me3 modification to that of thedemethylase; and

(3) a gene encoding a polypeptide that has 90% (or 92%, 94%, 96%, 98%,or 99%) or more homology to the amino acid sequence of any one or moreof the enzymes described above and has a substantially equivalent actionof substantially removing or reducing H3K27me3 modification to that ofthe demethylase.

An enzyme having a mutation may be a naturally occurring one, or may beone obtained by introducing a mutation on the basis of a gene of naturalorigin. Means for introducing a mutation is known per se, and forexample, a site-directed mutagenesis method, a homologous generecombination method, a primer extension method, a polymerase chainreaction (hereinafter abbreviated as PCR), and the like may be usedalone or in combination thereof as appropriate.

The method may be performed in conformity with any of methods describedin the literatures (“Molecular Cloning: A Laboratory Manual, secondedition” edited by Sambrook et al., 1989, Cold Spring Harbor Laboratory;and “Lab Manual: Genetic Engineering” edited by Masami Muramatsu, 1988,Maruzen), or by modifying these methods, and Ulmer's technology (Ulmer,K. M., “Science”, 1983, volume 219, p. 666-671) may also be utilized. Inthe case of a peptide, from the viewpoint of preventing alteration ofbasic properties of the peptide (e.g., physical properties, function,physiological activity, or immunological activity) in the introductionof a mutation, for example, mutual substitution between homologous aminoacids (e.g., polar amino acids, non-polar amino acids, hydrophobic aminoacids, hydrophilic amino acids, positively charged amino acids,negatively charged amino acids, and aromatic amino acids) is easilyconceivable.

(JMJD3)

JMJD3 is known as a demethylase for H3K27me3 of a histone (mouseNP_001017426, human NP_001073893), and even in its full length(NP_001073893, SEQ ID NO: 3), has an action of substantially removing orreducing the H3K27me3 modification of pluripotent stem cells.Surprisingly, the inventors of the present invention have confirmed thatJMJD3c having the JmjC domain {SEQ ID NO: 4, catalytic domain: SEQ IDNO: 5 (amino acids 1376-1484)} has a stronger action of substantiallyremoving or reducing H3K27me3 modification as compared to full-lengthJMJD3.

A preferred base sequence of JMJD3 is a base sequence set forth in SEQID NO: 32.

In addition, the JMJD3 of the present invention includes the followingembodiments as well:

(1) a protected derivative, sugar chain-modified derivative, acylatedderivative, or acetylated derivative of an amino acid sequence set forthin SEQ ID NO: 3;

(2) an amino acid sequence that has 90% (or 92%, 94%, 96%, 98%, or 99%)or more homology to the amino acid sequence set forth in SEQ ID NO: 3and has a substantially equivalent action of substantially removing orreducing H3K27me3 modification to that of the JMJD3;

(3) an amino acid sequence that has 100 to 10, 50 to 30, 40 to 20, 10 to5, or 5 to 1 amino acid substituted, deleted, inserted, and/or added inthe amino acid sequence set forth in SEQ ID NO: 3 and has asubstantially equivalent action of substantially removing or reducingH3K27me3 modification to that of the JMJD3;

(4) a protected derivative, sugar chain-modified derivative, acylatedderivative, or acetylated derivative of an amino acid sequence set forthin SEQ ID NO: 4;

(5) an amino acid sequence that has 90% (or 92%, 94%, 96%, 98%, or 99%)or more homology to the amino acid sequence set forth in SEQ ID NO: 4and has a substantially equivalent action of substantially removing orreducing H3K27me3 modification to that of the JMJD3c;

(6) an amino acid sequence that has 100 to 10, 50 to 30, 40 to 20, 10 to5, or 5 to 1 amino acid substituted, deleted, inserted, and/or added inthe amino acid sequence set forth in SEQ ID NO: 4 and has asubstantially equivalent action of substantially removing or reducingH3K27me3 modification to that of the JMJD3c; and

(7) an amino acid sequence that includes the amino acid sequence setforth in SEQ ID NO: 5 and has a substantially equivalent action ofsubstantially removing or reducing H3K27me3 modification to the JMJD3c.

It is appropriate that the “sequence homology” be generally 70% or more,preferably 80%, more preferably 85% or more, still more preferably 90%or more, even more preferably 95% or more, most preferably 98% or moreof an entire amino acid sequence.

In addition, the JMJD3 gene of the present invention includes thefollowing:

(1) a gene encoding a polypeptide consisted of an amino acid sequenceset forth in any one of SEQ ID NOS: 3 to 5;

(2) a gene encoding a polypeptide that has 1 to 20 (or 1 to 15, 1 to 10,1 to 7, 1 to 5, or 1 to 3) amino acids substituted, deleted, inserted,and/or added in the amino acid sequence set forth in any one of SEQ IDNOS: 3 to 5 and has a substantially equivalent action of substantiallyremoving or reducing H3K27me3 modification to that of the amino acidsequence set forth in any one of SEQ ID NOS: 3 to 5;

(3) a gene encoding a polypeptide that has 90% (or 92%, 94%, 96%, 98%,or 99%) or more homology to the amino acid sequence set forth in any oneof SEQ ID NOS: 3 to 5 and has a substantially equivalent action ofsubstantially removing or reducing H3K27me3 modification to that of theamino acid sequence set forth in any one of SEQ ID NOS: 3 to 5;

(4) a gene consisted of a base sequence set forth in any one of SEQ IDNOS: 6 to 8 and 32;

(5) a gene encoding a polypeptide that hybridizes with a base sequencecomplementary to the base sequence set forth in any one of SEQ ID NOS: 6to 8 and 32 under stringent conditions and has a substantiallyequivalent action of substantially removing or reducing H3K27me3modification to that of the amino acid sequence set forth in any one ofSEQ ID NOS: 3 to 5;

(6) a gene that has a sequence of 1 to 50 (or 1 to 40, 1 to 30, 1 to 20,1 to 15, 1 to 10, 1 to 5, or 1 to 3 bases substituted, deleted,inserted, and/or added in the DNA consisted of the base sequence setforth in any one of SEQ ID NOS: 6 to 8 and 32; and

(7) a gene having 90% (or 92%, 94%, 96%, 98%, or 99%) or more homologyto the gene formed of the base sequence set forth in any one of SEQ IDNOS: 6 to 8 and 32.

(Transcription Factor Required for Highly Efficient Induction ofDifferentiation into Desired Cell Type)

The embodiment of the “transcription factor required for highlyefficient induction of differentiation into the desired cell type” to beused in the method of the present invention is not particularly limited,but examples thereof may include, but not particularly limited to,nucleic acids, such as RNA and DNA, synthetic nucleic acids, andproteins.

In addition, in the method of the present invention, examples of thedesired cell type may include a skeletal muscle, the liver(hepatocytes), neurons, chondrocytes, bone cells, and hematopoieticcells.

{Transcription Factor required for Induction of Differentiation intoSkeletal Muscle (in particular, Cells present in Skeletal Muscle)}

A method of inducing differentiation into a skeletal muscle is asdescribed below.

A single transcription factor, or two or more transcription factorsselected from the group of MYOD1, NRF1, SALL4, ZIC1, KLF9, ZNF281, CTCF,HES1, HOXA2, TBX5, TP73, ERG, MAB21L3, PRDM1, NFIC, CTCFL, FOXP1, HEY1,PITX2, JUNB, KLF4, ESX1, TFAP2C, FOS, TFE3, FOSL1, GRHL2, TBX2, NFIB,and IRF4 are introduced into pluripotent stem cells. In particular,MYOD1 is preferably introduced into pluripotent stem cells.

{Transcription Factor Required for Induction of Differentiation intoLiver (in Particular, Cells Present in Liver, i.e., Hepatoblasts)}

A method of inducing differentiation into the liver (in particular, theliver or the fetal liver) is as described below.

Liver: A single transcription factor, or two or more transcriptionfactors selected from HNF1A, TCF-1, SALL4, TGIF1, MAB21L3, ZIC1, EGFLAM,PITX2, HNF4A, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6, and TCF4 areintroduced into human pluripotent stem cells.

Fetal liver: A single transcription factor, or two or more transcriptionfactors selected from HNF1A, TCF-1, SIX5, HNF4A, SIN3A, ID1, and HNF1Aare introduced into human pluripotent stem cells.

In particular, HNF1A is preferably introduced into pluripotent stemcells.

(Transcription Factor Required for Induction of Differentiation intoNeurons)

A method of inducing differentiation into neurons is as described below.

A single transcription factor, or two or more, three or more, four ormore, or five transcription factors selected from NEUROG1, NEUROG2,NEUROG3, NEUROD1, and NEUROD2 are introduced into human pluripotent stemcells.

(Method of Introducing Target Gene into Genome of Pluripotent Stem Cell)

In a step of the method of the present invention, a method known per semay be used without any particular limitation as a method of introducingthe gene for the compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein (or the gene for thecompound having an action of substantially removing or reducing H3K27me3modification) and/or the transcription factor required for highlyefficient induction of differentiation into the desired cell type intothe genome of the pluripotent stem cell. There may be preferably used anexpression cassette inserted between PiggyBac transposase recognitionsequences (PB sequences) developed by Woltjen et al. (reference: Nature458: 766-770, 2009.), which is a mechanism by which a gene to beintroduced is actively incorporated into pluripotent stem cells (inparticular, the genome of human ES cells). The expression cassette is asystem capable of efficiently establishing a genetically modifiedpluripotent stem cell line by introducing a drug selection cassette (seeFIG. 5).

(Method of Introducing Target Protein into Pluripotent Stem Cell)

In a step of the method of the present invention, a method known per semay be used as a method of introducing the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the compound having an action of substantially removing orreducing H3K27me3 modification) (in particular, protein) and/or thetranscription factor (protein) required for highly efficient inductionof differentiation into the desired cell type into the genome of thepluripotent stem cell, and examples thereof may include: a methodinvolving using a protein transfection reagent; a method involving usinga fusion protein having added thereto a cell-penetrating peptide; and amicroinjection method.

The “cell-penetrating peptide” of the present invention is a peptidehaving a property of migrating into a cell, more specifically a propertyof penetrating a cell membrane, still more specifically a property ofpenetrating a cell membrane or a nuclear membrane to penetrate intocytoplasm or a nucleus. The amino acid sequence of the peptide is notparticularly limited, but examples thereof may include TAT(GRKKRRQRRRPQ: SEQ ID NO: 9), r8 {rrrrrrrr (D-form-R): SEQ ID NO: 10},and MPG-8 (βAFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 11).

The target protein encompasses both of the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the compound having an action of substantially removing orreducing H3K27me3 modification) (in particular, protein) and/or thetranscription factor (protein) required for highly efficient inductionof differentiation into the desired cell type.

(Gene Knockout Method)

A gene knockout method is available as a method other than theforegoing. A “pluripotent stem cell in which a POU5F1 gene has beendisrupted” may be generated by the gene knockout method. The“pluripotent stem cell in which a POU5F1 gene has been disrupted” meansthat normal expression of the POU5F1 gene is inhibited due to artificialmodification of the sequence of a POU5F1 gene region, and as a result,the expression of POU5F1 is suppressed and a POU5F1 protein is notnormally expressed.

In addition, the “whole” in “modification or deletion of part or thewhole of the POU5F1 gene” refers to the protein-coding region of POU5F1genomic DNA.

In addition, the “part” refers to a region that is part of theprotein-coding region and that has a length required for inhibitingnormal expression of the POU5F1 gene.

Further, the “modification” refers to modification of the base sequenceof a target region in genomic DNA into another base sequence bysubstituting, deleting, inserting, and/or adding a single nucleotide ortwo or more of nucleotides.

(Differentiation Induction Kit for Inducing Differentiation ofPluripotent Stem Cell into Desired Cell Type with High Efficiency)

A differentiation induction kit for inducing differentiation of apluripotent stem cell into a desired cell type with high efficiency ofthe present invention (hereinafter sometimes referred to as “kit of thepresent invention”) includes any one or more of the following modes.

(1) Pluripotent Stem Cell in which Expression Amount of POU5F1 Proteinhas been substantially removed or reduced and/or H3K27me3 Modificationhas been substantially removed or reduced

A pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced and/or H3K27me3modification has been substantially removed or reduced can be easilygenerated by the method of the present invention described above.

A implementer can easily induce differentiation into the desired celltype by introducing the transcription factor required for induction ofdifferentiation into the desired cell type as described above into thepluripotent stem cell.

In addition, such pluripotent stem cell encompasses a pluripotent stemcell having a gene construct inducible with doxycycline or the likeinserted into the genome thereof so that a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein, a demethylase, or the like can be transiently forciblyexpressed therein.

(2) Gene for Compound having Action of substantially removing orreducing Expression Amount of POU5F1 Protein and/or Demethylase Gene forKit of the Present Invention

The implementer can easily generate the pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and/or H3K27me3 modification has been substantially removedor reduced by adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and/or a demethylase gene for a kit to a pluripotent stem cell.

Examples of the anti-POU5F1 antibody gene for a kit may include, but notparticularly limited to, commercially available antibody genes.

Examples of the demethylase gene for a kit may include, but notparticularly limited to, mRNAs, DNAs, and proteins of demethylase genes(e.g., JMJD3c).

(3) Gene for Compound having Action of substantially removing orreducing Expression Amount of POU5F1 Protein and/or Demethylase Gene,and Gene containing Transcription Factor required for Induction ofDifferentiation into Desired Cell Type for Kit of the Present Invention

The implementer can easily generate the pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and/or H3K27me3 modification has been substantially removedor reduced, and further, can induce differentiation thereof into thedesired cell type with high efficiency by adding a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and/or a demethylase gene, and a genecontaining a transcription factor required for induction ofdifferentiation into the desired cell type for a kit to a pluripotentstem cell.

Those genes may be present on one gene, or on separate genes. When thegenes are present on separate genes, the gene for the compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein (or demethylase gene) and the transcription factorrequired for induction of differentiation into the desired cell type maybe added to the pluripotent stem cell simultaneously or at separatetimes.

(4) Compound having Action of substantially removing or reducingExpression Amount of POU5F1 Protein and/or Demethylase for Kit of thePresent Invention

The implementer can easily generate the pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and/or H3K27me3 modification has been substantially removedor reduced by adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein and/or ademethylase for a kit to a pluripotent stem cell.

(5) Gene Construct carrying Gene for Compound having Action ofsubstantially removing or reducing Expression Amount of POU5F1 Proteinand/or Demethylase Gene

The user can easily generate the pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and/or H3K27me3 modification has been substantially removed orreduced by introducing a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and/or a demethylase gene into the genome ofa pluripotent stem cell.

The gene construct may contain a promoter sequence, a geneexpression-enhancing sequence, a marker gene, a reporter sequence, adrug resistance gene, and the like as required in addition to the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein and/or the demethylase gene.

(6) Gene Construct carrying: Gene for Compound having Action ofsubstantially removing or reducing ExpressionAmount of POU5F1 Proteinand/or Demethylase Gene; and Transcription Factor required for Inductionof Differentiation into Desired Cell Type

The implementer can easily generate the pluripotent stem cell in whichan expression amount of a POU5F1 protein has been substantially removedor reduced and/or H3K27me3 modification has been substantially removedor reduced, and further, can induce differentiation thereof into thedesired cell type by introducing a gene construct carrying: a gene for acompound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein and/or a demethylase gene; and atranscription factor required for induction of differentiation into thedesired cell type into the genome of a pluripotent stem cell.

Those genes may be present on one gene, or on separate genes. When thegenes are present on separate genes, the gene for the compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein and/or the demethylase gene, and the transcription factorrequired for induction of differentiation into the desired cell type maybe expressed in the genome of the pluripotent stem cell simultaneouslyor at separate times.

The gene construct may contain a promoter sequence, a geneexpression-enhancing sequence, a marker gene, a reporter sequence, adrug resistance gene, and the like as required in addition to the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein and/or the demethylase gene,and the transcription factor required for induction of differentiationinto the desired cell type.

(Proteins associated with Undifferentiated State Maintenance other thanPOU5F1) NANOG, SOX2, SOX3, KLF2, KLF4, KLF5, TBX3, ESRRB, SALL4, STAT3,ZIC3, LIN28, and TCF3 are known as proteins associated withundifferentiated state maintenance. Accordingly, it is considered thatpluripotent stem cells in which expression amounts of the proteins ofNANOG, SOX2, SOX3, KLF2, KLF4, KLF5, TBX3, ESRRB, SALL4, STAT3, ZIC3,LIN28, and TCF3 have been substantially removed or reduced can bedifferentiated into the desired cell type with high efficiency.

Further, a pluripotent stem cell in which an expression amount of anyone or more of the above-mentioned proteins has been substantiallyremoved or reduced and an expression amount of a POU5F1 protein has beensubstantially removed or reduced (pluripotent stem cell in which anexpression amount of any one or more of the above-mentioned proteins hasbeen substantially removed or reduced and an expression amount of aPOU5F1 protein has been substantially removed or reduced, and which hasa histone in which H3K27me3 modification has been substantially removedor reduced) is also included in the present invention.

In addition, a method of differentiating a pluripotent stem cell into adesired cell type including a step of adding a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of any one or more of the above-mentioned proteins, a gene for acompound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein (further, a demethylase gene orthe like), and a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell isalso encompassed in the present invention.

A method of differentiating a pluripotent stem cell into a desired celltype of the present disclosure may be exemplified by, but notparticularly limited to, a method including any one of the followingsteps (1) to (9):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a transcription factor gene required forinduction of differentiation into the desired cell type, into a genomeof a pluripotent stem cell;

(3) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a gene construct carrying a transcriptionfactor required for induction of differentiation into the desired celltype, into a genome of a pluripotent stem cell;

(4) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell inwhich an expression amount of a POU5F1 protein has been substantiallyremoved or reduced;

(5) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell inwhich a compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein is forcibly expressed;

(6) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein and atranscription factor required for differentiation into the desired celltype to a pluripotent stem cell;

(7) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell inwhich a POU5F1 gene has been disrupted;

(8) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and a transcription factor required forinduction of differentiation into the desired cell type to a pluripotentstem cell; and

(9) a step of adding a transcription factor required for induction ofdifferentiation into the desired cell type to a pluripotent stem cell inwhich an expression amount of a POU5F1 protein has been substantiallyremoved or reduced and which has a histone in which H3K27me3modification has been substantially removed or reduced.

The present disclosure also includes any one of the followingpluripotent stem cells for differentiation into a desired cell type:

(1) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(2) a pluripotent stem cell for differentiation into a desired celltype, in which a gene for a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein isforcibly expressed;

(3) a pluripotent stem cell for differentiation into a desired celltype, which has a gene construct carrying a gene for a compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein inserted into the genome thereof;

(4) a pluripotent stem cell for differentiation into a desired celltype, in which a POU5F1 gene has been disrupted; and

(5) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

The present disclosure also includes a use of any one of the followingpluripotent stem cells for differentiation into a desired cell type:

(1) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(2) a pluripotent stem cell for differentiation into a desired celltype, in which a gene for a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein isforcibly expressed;

(3) a pluripotent stem cell for differentiation into a desired celltype, which has a gene construct carrying a gene for a compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein inserted into the genome thereof;

(4) a pluripotent stem cell for differentiation into a desired celltype, in which a POU5F1 gene has been disrupted; and

(5) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

The present disclosure also includes a use of any one of the followingpluripotent stem cells for differentiation into a desired cell type, inproduction of a differentiation induction kit for differentiating apluripotent stem cell into a desired cell type:

(1) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(2) a pluripotent stem cell for differentiation into a desired celltype, in which a gene for a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein isforcibly expressed;

(3) a pluripotent stem cell for differentiation into a desired celltype, which has a gene construct carrying a gene for a compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein inserted into the genome thereof;

(4) a pluripotent stem cell for differentiation into a desired celltype, in which a POU5F1 gene has been disrupted; and

(5) a pluripotent stem cell for differentiation into a desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

The present invention is more embodied described below by Examples.However, the present invention is not limited to these Examples. All ofthese Examples have been approved by the Ethics Committee of KeioUniversity School of Medicine.

Example 1

(Materials and Methods)

Examples 2 to 6 were carried out using materials and methods describedbelow. The details are as described below.

(Human Pluripotent Stem Cell Culture and Differentiation InductionMethods)

Human ES cell (hESC) lines SEES-3 and H9 were obtained from the NationalCenter for Child Health and Development (National Research Institute forChild Health and Development) and the Cell Research Institute, USA,respectively. A human induced pluripotent stem cell (hiPSC) line wasobtained from RIKEN or the Center for iPS Cell Research and Application,Kyoto University. hESC/iPSCs were cultured using StemFit AK-03 medium(Ajinomoto) on iMatrix-511 (Nippi)-coated plates without use of feedercells. A ROCK (Rho-associated coiled-coil forming kinase/Rho-associatedkinase) inhibitor Y-27632 was added to the medium during cell subculturein order to inhibit apoptosis induced by cell detachment during cellpassaging.

For myogenic differentiation, the hESC/iPSCs were cultured in a mediumof αMEM (Gibco) supplemented with 5% KSR, 1 mM sodium pyruvate, 0.1 mMnon-essential amino acids, 2 mM glutamine, 0.1 mM β-mercaptoethanol, andpenicillin/streptomycin (50 U/50 μg/ml) on iMatrix-511-coated plates.

For albumin secretion hepatocyte differentiation, the hESC/iPSCs werecultured in the above-mentioned medium on Matrigel (BD)-coated plates,then cultured in RPMI 1640 medium supplemented with 1 mM NaB, 100 ng/mlActivin A, 50 ng/ml Wnt3a, 1×B27, and 2 mM GlutaMAX for 1 day,subsequently cultured in DMEM medium supplemented with 1% DMSO, 0.5 mMMTG, 1% NEAA, 1 mM GlutaMAX, and 20% KSR for 5 days, and finallycultured in HCM medium (Lonza) supplemented with 20 ng/ml HGF and 20ng/ml Oncostatin M for 7 days.

(siRNA Transfection)

siRNA against POU5F1 (sense strand: GCCCGAAAGAGAAAGCGAATT: SEQ ID NO:12, antisense strand: UUCGCUUUCUCUUUCGGGCCT: SEQ ID NO: 13) identical tothat used in the literature “Proceeding of national academy of sciences109, 4485-4490 (2012)” was purchased from Applied Biosystems and used(product number: s10873). siRNA serving as a negative control was alsopurchased from Applied Biosystems and used. siRNA transfections wereperformed with Lipofectamine Messenger Max (Invitrogen), according tothe instructions of the accompanying manual. The B18R interferoninhibitor (eBioscience) was added to the culture medium to increase theviability of the transfected cells. The medium was replaced 2 hours to 3hours after each transfection.

(Modified mRNA Synthesis and Transfection)

The protein-coding regions (Open Reading Frames, ORFs) of a redfluorescent protein mCherry, a green fluorescent protein Emerald, andtissue-specific transcription factors {MYOD1 (SEQ ID NO: 26), HNF1A (SEQID NO: 27), RUNX2 (SEQ ID NO: 28), SOX9 (SEQ ID NO: 29), SPI1 (SEQ IDNO: 30), and ASCL1 (SEQ ID NO: 31)} were subcloned into a pCRIIconstruct containing the 5′ UTR and 3′ UTR of mouse α-globin, whichincreased mRNA stability and translation efficiency, to preparetemplates used to synthesize mRNAs.

Modified mRNAs were synthesized on the basis of the description of theliterature“Cell stem cell 7, 618-630 (2010)”. Briefly speaking, a T7promoter and a poly (A) tail were added through PCR reaction using aKAPA taq kit (Kapa Biosystems). RNAs were synthesized from PCR productsusing a MEGAscript T7 kit (Ambion) together with ARCA cap analog (NewEngland Biolabs), ATP, GTP, 5-Methyl-CTP (TriLink), and pseudo-UTP(TriLink). The synthetic mRNAs were purified using a MEGAclear kit(Ambion). Synthetic mRNA transfections were performed with LipofectamineMessengerMax (Invitrogen) according to the instructions of theaccompanying manual. The B18R interferon inhibitor (eBioscience) wasadded to the culture medium to increase the viability of the transfectedcells. The medium was replaced 2 hours to 3 hours after eachtransfection.

(Antibody)

The following antibodies were used:

POU5F1 (Santa Cruz, sc-5279);

β-ACTIN (Cell Signaling, 4970S);

MyHC (R&D MAB4470);

ALBUMIN (Abcam ab10241); and

AFP (R&D MAB1368).

(Immunostaining)

The cells were fixed in 4% PFA for 10 minutes at room temperature andpermeabilized in 0.5% Triton-X-containing PBS for 10 minutes. The cellswere treated a blocking in 2% BSA-containing PBS for 10 minutes, andcultured with primary antibodies in a blocking solution (1:500) for from2 hours to 3 hours at room temperature or overnight at 4° C. The cellswere washed twice in PBS, and then cultured with Alexa dye-conjugatedsecondary antibodies (Invitrogen) in a blocking solution (1:500) for 1hour at room temperature. Nuclei were counterstained with DAPI (Dako).Immunofluorescence was visualized with an inverted fluorescencemicroscope IX73 (Olympus). Images were obtained using Olympus cellSensimaging software.

(Immunoblotting Method)

The cells were lysed with a sample buffer (50 mM Tris-HCl, pH 6.8, 2%SDS, 6% 2-mercaptoethanol, and 500 mg/ml urea). The proteins wereseparated by SDS-PAGE using a 4-15% polyacrylamide gel (Biorad) and wereelectrically transferred to polyvinylidene fluoride membranes (Biorad).The membranes were blocked for 1 hour in 0.1% Tween-20-containingTris-buffered saline (TBST) and 5% skimmed milk. The membranes werewashed in TBST and then incubated with primary antibodies in 2%BSA-containing TBS (1:1,000 dilution) for from 2 hours to 3 hours atroom temperature or overnight at 4° C. The membranes were washed andincubated with horseradish peroxidase-conjugated secondary antibodies(GE) for 1 hour at room temperature. The membranes were washed in TBST,and immunoreactivity was visualized using ECL Prime Detection Kit (GE)and detected using Luminescent Image Analyzer (LAS-4000; Fujifilm).

(qRT-PCR)

Total RNA was isolated with TRIzol reagent (Invitrogen), and cDNAs weregenerated with random hexamers using a ReverTra Ace kit (Toyobo).Real-time PCR was performed using a SYBR Green PCR system (Takara). Theprimer sequences used for RT-PCR are listed below.

MYOG primer (Forward): (SEQ ID NO: 14) gccagactatccccttcctcMYOG primer (Reverse): (SEQ ID NO: 15) gaggccgcgttatgataaaaAFP primer (Forward): (SEQ ID NO: 16) tgggacccgaactttccaAFP primer (Reverse): (SEQ ID NO: 17) ggccacatccaggactagtttcCOL1A1 primer (Forward): (SEQ ID NO: 18) cctggatgccatcaaagtctCOL1A1 primer (Reverse): (SEQ ID NO: 19) tcttgtccttggggttcttgCOL2A1 primer (Forward): (SEQ ID NO: 20) tttcccaggtcaagatggtcCOL2A1 primer (Reverse): (SEQ ID NO: 21) cttcagcacctgtctcaccaCD45 primer (Forward): (SEQ ID NO: 22) tcctggactcccaaaatctgCD45 primer (Reverse): (SEQ ID NO: 23) accttgaacccgaacatgagNESTIN primer (Forward): (SEQ ID NO: 24) tggttttccagagtcttcagtgaNESTIN primer (Reverse): (SEQ ID NO: 25) gaaacagccatagagggcaaa

Example 2

(Pluripotent Stem Cells in which Expression Amount of POU5F1 Protein hasbeen Reduced)

In this Example, it was confirmed whether pluripotent stem cells inwhich the expression amount of a POU5F1 protein had been forciblyreduced were able to be generated or not. The details are as describedbelow.

(Confirmation of Suppression of Expression of POU5F1 by siRNATransfection)

Effects of siRNA (siPOU5F1) on POU5F1 in human ES cells were confirmedby an immunoblotting method and an immunostaining method (FIG. 6). Cellson Day 3 after siPOU5F1 transfection were analyzed, and as a result, itwas confirmed that the POU5F1 protein was significantly decreased ascompared to cells transfected with negative control siRNA (scramblesiRNA sequence against POU5F1: siControl) (FIG. 6). The siPOU5F1transfection decreased an NANOG protein serving as a molecular markerfor pluripotent stem cells.

Thus, the disappearance of pluripotency by the siPOU5F1 transfection wasconfirmed.

(Confirmation of Morphological Change of Human ES Cells by siPOU5F1Transfection)

Human ES cells have small round shapes when their undifferentiated stateis maintained. It was confirmed that cells underwent a morphologicalchange into flat shapes when transfected with siPOU5F1, and weredirected toward differentiation through POU5F1 suppression (FIG. 7).

As this Example's result, it was confirmed that pluripotent stem cellsin which the expression amount of the POU5F1 protein had been reducedwere able to be generated, and that the cells were promoted towarddifferentiation.

Example 3

(Confirmation of Induction of Differentiation of Pluripotent Stem Cellsin which Expression Amount of POU5F1 Protein has been Reduced intoDesired Cell Types)

In this Example, it was confirmed whether differentiation into desiredcell types was induced by introducing (adding) transcription factorsrequired for induction of differentiation into the desired cell types topluripotent stem cells in which the expression amount of the POU5F1protein had been reduced. The details are as described below.

siPOU5F1 and modified synthetic RNAs (synRNAs) for a tissue-specifictranscription factors were introduced into human ES cells, and theexpression of differentiation marker genes MYOG (skeletal muscles), AFP(hepatocytes), COL1A1 (bone cells), COL2A1 (cartilage), CD45(hematopoietic cells), and NESTIN (nerves) was examined by a real-timeRT-PCR method.

As the tissue-specific transcription factors, MYOD1 (skeletal muscles),HNF1A (liver), RUNX2 (bone cells), SOX9 (cartilage), SPI1 (blood), andASCL1 (nerves) were used. As a negative control, synRNA for mCherry orEmerald was introduced.

siControl or siPOU5F1 and synRNA were simultaneously introduced (added)to the cells, and 1 day after that, the synRNA was further introducedthereto twice. The next day (2 days after the first introduction), thecells were sampled and analyzed. The medium used was a medium forundifferentiated state maintenance. As a result, it was confirmed that,when siPOU5F1 was introduced in combination with the transcriptionfactors, the expression of the differentiation markers for therespective tissues was significantly increased (FIG. 8). While, assiControl or the transcription factors were introduced alone, anyincreases in expression of the differentiation markers were hardlydetected (FIG. 8).

As this Example's result, it was confirmed that differentiation intodesired cell types was efficiently induced by introducing (adding) thetranscription factors required for induction of differentiation into thedesired cell types to the pluripotent stem cells in which the expressionamount of the POU5F1 protein had been reduced.

Example 4

(Confirmation of Differentiation of Pluripotent StemCells in whichExpression Amount of POU5F1 Protein has been Reduced into Desired CellTypes)

In this Example, as examples of induction of differentiation intodesired cell types, a myogenic differentiation (skeletal muscle celldifferentiation) model using a myogenesis-regulating mastertranscription factor MYOD1, and a hepatocyte differentiation model usinghepatocyte nuclear factor-1-alpha (HNF1A) were adopted. It is known thatforced expression of MYOD1 alone cannot cause sufficient epigeneticchanges and transcriptional changes in hESCs, resulting in poor myogenicconversion (see Cell reports 3, 661-670 (2013)).

(Skeletal Muscle Cell Differentiation)

Human ES cells were co-transfected with siControl or siPOU5F1 and synRNAfor MYOD1 (MYOD1-synRNA), and 1 day after that, were further transfectedtwice with MYOD1-synRNA. In this case, the cells were cultured in askeletal muscle differentiation medium.

Two days after the last synRNA transfection, which was performed daysafter the first transfection, the cells were fixed and immunostained.The ES cells transfected with siControl hardly underwent a morphologicalchange even when transfected with MYOD1-synRNA, and few of the cellsexpressed Myosin Heavy Chain (MyHC) serving as a terminaldifferentiation marker for a skeletal muscle (FIG. 9).

However, as the ES cells transfected with siPOU5F1 were used, 2 daysafter the last MYOD1-synRNA transfection, fibrous muscle cells appearedat a high frequency and most of the cells were MyHC-positive, confirmingthat the cells were differentiated into skeletal muscle cells (FIG. 9).

In addition, similar effects were also able to be obtained in another EScell line (H9 hES cell) and a plurality of iPS cell lines (201B7 iPScell, 409B2 iPS cell, RIKEN-1A iPS cell, and tkDA3-4 iPS cell) (FIG.10).

(Hepatocyte Differentiation)

Human ES cells were co-transfected with siControl or siPOU5F1 and synRNAfor HNF1A (HNF1A-synRNA), and 1 day after that, were further transfectedtwice with HNF1A-synRNA. From the day after the last synRNAtransfection, which was performed 2 days after the first transfection,the cells were cultured in a hepatocyte differentiation medium, and 13days later, the cells were fixed and immunostained. While, the ES cellstransfected with siControl were transfected with HNF1A-synRNA, cellsexpressing AFP and albumin (ALB) serving as differentiation markers forhepatocytes were hardly observed (FIG. 11). Beside, in the case of thecells co-transfected with siPOU5F1 and HNF1A-synRNA, many cellsexpressing albumin and AFP were observed, and thus it was confirmed thatthe cells were differentiated into hepatocytes (FIG. 11).

As this Example's result, it was confirmed that differentiation intodesired cell types was able to be efficiently induced by introducing thetranscription factors required for induction of differentiation into thedesired cell types into the pluripotent stem cells in which theexpression amount of the POU5F1 protein had been reduced.

Example 5

(Confirmation of Induction of Differentiation of Pluripotent Stem Cellsin which Expression Amount of POU5F1 Protein has been Reduced andDemethylase is Forcibly Expressed into Desired Cell Types)

The inventors of the present invention have confirmed that pluripotentstem cells in which a demethylase is forcibly expressed allow thedifferentiation resistance of human pluripotent stem cells to disappear,thereby facilitating differentiation induction. A molecular mechanism bywhich the expression amount of the POU5F1 protein is reduced isdifferent from a molecular mechanism by which the demethylase isforcibly expressed.

In view of the foregoing, it was confirmed whether differentiation intodesired cell types was induced by introducing (adding) transcriptionfactors required for induction of differentiation into the desired celltypes to pluripotent stem cells in which the expression amount of thePOU5F1 protein had been reduced and the demethylase was forciblyexpressed, which were obtained by combining the reduction of theexpression amount of the POU5F1 protein and the forced expression of thedemethylase. The details are as described below.

Differentiation into the desired cell types was induced using synRNAsfor tissue-specific transcription factors MYOD1, HNF1A, SOX9, and SPI1.

It was confirmed that the expression of differentiation marker genesMYOG (skeletal muscle), AFP (hepatocyte), COL2A1 (cartilage), CD45(hematopoietic cell) was more increased in a synergistic manner in thecase of differentiation performed by combining the reduction of theexpression amount of the POU5F1 protein and the forced expression of thedemethylase than in the case of differentiation performed by only one ofthe reduction of the expression amount of the POU5F1 protein or theforced expression of the demetylase, and the expression with synRNA(FIG. 12).

More specifically, an about 18.2-fold increase in expression of MYOG, anabout 5.2-fold increase in expression of AFP, an about 2.8-fold increasein expression of COL2A1, and an about 2.2-fold increase in expression ofCD45 were achieved by combining the reduction of the expression amountof the POU5F1 protein and the forced expression of the demethylase ascompared to the case in which the expression amount of the POU5F1protein was only reduced.

Further, the combination of the reduction of the expression amount ofthe POU5F1 protein and the forced expression of the demethylase achievedan about 4.3-fold increase in expression of MYOG, an about 54.0-foldincrease in expression of AFP, an about 2.8-fold increase in expressionof COL2A1, and an about 4.5-fold increase in expression of CD45 ascompared to the case in which the demethylase was forcibly expressed.

In addition, in hepatocyte differentiation induction, the reduction ofthe expression amount of the POU5F1 protein achieved expression about10.4 times as high as that achieved by the forced expression of thedemethylase. Thus, concerning hepatocyte differentiation induction, thereduction of the expression amount of the POU5F1 protein showed asignificant effect as compared to the forced expression of thedemethylase.

As this Example's result, it was confirmed that the pluripotent stemcells in which the expression amount of the POU5F1 protein had beenreduced and the demethylase was forcibly expressed had a synergisticdifferentiation induction effect (advantageous effect) as compared tothe pluripotent stem cells in which the expression amount of the POU5F1protein had been reduced and the pluripotent stem cells in which thedemethylase was forcibly expressed.

Example 6

(Examples of Differentiation into Desired Cell Types Using PluripotentStem Cells of the Present Disclosure)

In this Example, differentiation into various desired cell types wasconfirmed using pluripotent stem cells in which the expression amount ofthe POU5F1 protein had been substantially removed or reduced on thebasis of the example described as Example 3.

(Differentiation into Skeletal Muscle Cells)

During 4-day culture, human pluripotent stem cells were co-transfectedwith siPOU5F1 and a MYOD1 gene (SEQ ID NO: 33, SEQ ID NO: 34) once, andthen transfected with the MYOD1 gene three times. It was confirmed thatthe cells were differentiated into skeletal muscle cells through the4-day culture.

(Differentiation into Hepatocytes)

During 12-day culture, human pluripotent stem cells were co-transfectedwith siPOU5F1 and a HNF1A gene (SEQ ID NO: 35, SEQ ID NO: 36) once, andthen transfected with the HNF1A gene twice. It was confirmed that thecells were differentiated into hepatocytes through the 12-day culture.

(Differentiation into Neurons)

During 5-day culture, human pluripotent stem cells are transfected withsiPOU5F1 once, and then transfected with a NEUROG1 gene (SEQ ID NO: 37,SEQ ID NO: 38), a NEUROG2 gene (SEQ ID NO: 39, SEQ ID NO: 40), a NEUROG3gene (SEQ ID NO: 41, SEQ ID NO: 42), a NEUROD1 gene (SEQ ID NO: 43, SEQID NO: 44), and a NEUROD2 gene (SEQ ID NO: 45, SEQ ID NO: 46) threetimes. The cells can be differentiated into neurons through the 5-dayculture.

Example 7

(Examples of Differentiation into Desired Cell Types Using PluripotentStem Cells of the Present Disclosure by Use of Sendai Virus Vector)

In this Example, unlike Example 3, differentiation into various desiredcell types is confirmed using pluripotent stem cells in which theexpression amount of the POU5F1 protein has been substantially removedor reduced, through use of a Sendai virus vector instead of syntheticmodified mRNA.

(Differentiation into Skeletal Muscle Cells)

A Sendai virus vector known per se (which is active at 33° C. and isinactivated at 37° C.) into which a transcription factor MYOD1 gene (SEQID NO: 33, SEQ ID NO: 34) has been cloned is used.

A suspension of human ES cells or iPS cells is infected with the Sendaivirus vector at a multiplicity of infection (MOI) of 1 to 100 (25).After that, the cells are transferred to a culture plate, and the cellsare cultured in a CO₂ incubator kept at 33° C. for 3 days. After that,the cells are transferred to a CO₂ incubator at 37° C., and culture iscontinued. As a result, skeletal muscle cells can be observed asdifferentiated cells.

(Differentiation into Hepatocytes)

A Sendai virus vector known per se into which a transcription factorHNF1A gene (SEQ ID NO: 35, SEQ ID NO: 36) has been cloned is used.

A suspension of human ES cells or iPS cells is infected with the Sendaivirus vector at a MOI of 1 to 100 (25). After that, the cells aretransferred to a culture plate, and the cells are cultured in a CO₂incubator kept at 33° C. for 3 days. After that, the cells aretransferred to a CO₂ incubator at 37° C., and culture is continued. As aresult, hepatocytes can be observed as differentiated cells.

(Differentiation into Neurons)

A Sendai virus vector known per se into which a transcription factorNEUROG3 gene (SEQ ID NO: 41, SEQ ID NO: 42) has been cloned is used.

A suspension of human ES cells or iPS cells is infected with the Sendaivirus vector at a MOI of 1 to 100 (25). After that, the cells aretransferred to a culture plate, and the cells are cultured in a CO₂incubator kept at 33° C. for 3 days. After that, the cells aretransferred to a CO₂ incubator at 37° C., and culture is continued. As aresult, neurons (motor neurons) can be observed as differentiated cells.

Conclusion

The inventors of the present invention have confirmed by theabove-mentioned Examples that the method of differentiating apluripotent stem cell into a desired cell type with high efficiency andthe differentiation induction kit for differentiating a pluripotent stemcell into a desired cell type with high efficiency of the presentinvention each have at least any one of the following effects.

(1) The period of time required for cell differentiation starting withthe pluripotent stem cell is shortened and/or the differentiationinduction efficiency is improved.(2) As modified synthetic mRNA for a gene is used to introduce the geneinto the pluripotent stem cell, the introduced gene is not incorporatedinto the genome of the pluripotent stem cell, with the result that thereis no risk of canceration or the like after cell differentiationinduction.(3) In the introduction of the gene into the pluripotent stem cell usingthe modified synthetic mRNA, the timing and number of times of theaddition of the mRNA for the gene can be easily changed, and henceoptimal conditions specific to each of various desired cell types can beselected so as to differentiate the pluripotent stem cell into thedesired cell types.(4) A method of reducing undifferentiated state maintenance of apluripotent stem cell and a method of reducing differentiationresistance thereof are combined with each other to shorten the period oftime required for cell differentiation starting with the pluripotentstem cell and to improve the differentiation induction efficiency in asynergistic manner.

INDUSTRIAL APPLICABILITY

According to the present invention, the novel method of differentiatinga pluripotent stem cell into a desired cell type with high efficiencycan be provided.

1-13. (canceled)
 14. A method of differentiating a pluripotent stem cellinto a desired cell type, comprising any one of the following steps (1)to (2): (1) a step of adding an siRNA, shRNA, or antisense RNA having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein, a demethylase gene, and a modified mRNA encoding atranscription factor required for induction of differentiation into thedesired cell type to a pluripotent stem cell; and (2) a step of adding amodified mRNA encoding a transcription factor required for induction ofdifferentiation into the desired cell type and an siRNA, shRNA, orantisense RNA having an action of substantially removing or reducing anexpression amount of a POU5F1 protein to a pluripotent stem cell whichhas a histone in which H3K27me3 modification has been substantiallyremoved or reduced, wherein the differentiation method is of thepluripotent stem cell into a hepatocyte, and the transcription factor ofeach of the steps (1) to (2) comprises HNF1A, and the removing orreducing of the expression amount of the POU5F1 protein of each of thesteps (1) to (2), and production of HNF1A protein of each of the steps(1) to (2) lead to the differentiation of the pluripotent stem cell andexpression of markers of hepatocyte differentiation.
 15. Thedifferentiation method according to claim 14, wherein the pluripotentstem cell is co-transfected with siPOU5F1 and synRNA for HNF1A.